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Multi-active-site fluorescent probe as well as synthesis method and application thereof

A technology of active sites and fluorescent probes, applied in the field of analytical chemistry, can solve difficult and difficult problems

Active Publication Date: 2020-08-21
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the similarity in structure and properties between Cys, Hcy and GSH, it is very difficult to effectively distinguish between two of them at the same time, and it is even more difficult to distinguish the three at the same time
At present, there are multi-fluorescent channel probes used to distinguish and detect Cys, Hcy and GSH at the same time (such as Chinese patent applications CN108484622A, CN108752331A, etc.), but few reports can exclude SO 2 interfering probe

Method used

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  • Multi-active-site fluorescent probe as well as synthesis method and application thereof
  • Multi-active-site fluorescent probe as well as synthesis method and application thereof
  • Multi-active-site fluorescent probe as well as synthesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]

[0034] (E)-3-(2-(2-(4-Chloro-7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-3,3-dimethyl yl-3H-benzo[g]indol-1-ium-1-yl)propane-1-sulfonate

[0035]7-(N,N-diethylamino)-4-chlorocoumarin-3-carbaldehyde (50.0 mg, 179 μmol) and 1,1,2-trimethyl-3-(3-sulfopropyl )-1H-benzo[E]indole internal salt (65.0mg, 197μmol) was dissolved in 10mL of absolute ethanol, 2 drops of acetic acid was added dropwise, under nitrogen protection, the reaction was stirred under reflux; after the reaction, the reaction solution was cooled to room temperature, Concentrated under reduced pressure, purified by silica gel column chromatography (v:v, dichloromethane:methanol=50:1), to obtain 71.0 mg (67% yield) blue-purple solid powder, which was identified as (E)-3-( 2-(2-(4-chloro-7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-3,3-dimethyl-3H-benzo[ g] Indol-1-ium-1-yl)propane-1-sulfonate.

[0036] Mp 176-179°C. 1 H NMR (300MHz, DMSO-d 6 )δ8.58–8.34(m,2H),8.34–8.08(m,4H),7.95–7.55(m,3H),7.01(d,...

Embodiment 2

[0038] Differential detection of Cys, Hcy, GSH and SO in in vitro environment with a multi-active-site fluorescent probe 2 Applications

[0039] Probes (1 mM) were dissolved in phosphate buffered saline (PBS, 50 mM, pH 7.4, containing 15% v / v acetonitrile) to prepare stock solutions. Solutions of various other analytes were prepared in deionized water. SO 2 The generating reagent is NaHSO 3 . Except for special instructions, the detection conditions of the ultraviolet fluorescence test are: dilute the stock solution with phosphate buffer (50mM, pH 7.4, containing 15% acetonitrile), add the corresponding detection substrate, and the test concentration of the probe is 10μM, at room temperature Incubate for 2 hours, Cys detection uses 365nm wavelength as excitation light, slit is 3nm / 3nm; Hcy detection uses 530nm wavelength as excitation light, slit is 5nm / 5nm; SO2 detection uses 600nm as excitation light, slit is 5nm / 5nm; GSH detection uses 650nm wavelength as the excitatio...

Embodiment 3

[0051] Multi-active site fluorescent molecular probe cytotoxicity test and fluorescence imaging test

[0052] Different biothiols have different levels in cells (Cys is 30-200μM, Hcy is 5-12μM, GSH is 1-10mM). According to relevant reports, the inventors selected human lung cancer cells A549 to conduct biothiol imaging experiments at the cellular level.

[0053] First, the MTT cytotoxicity test of the probe against A549 was carried out. Human lung cancer cells (A549) were grown in DMEM medium containing 10% (v / v) FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) at 37°C in a humid atmosphere. A549 cells were cultured with different concentrations of the probe (20 μM, 40 μM, 60 μM, 80 μM and 100 μM), and the cell viability results showed that the probe had negligible cytotoxicity to living cells ( Figure 14 ).

[0054] The experimental method for determining the intracellular imaging ability of probes to biothiols is as follows: A-E series first added 1mM N-ethylmalei...

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Abstract

The invention discloses a multi-active-site fluorescent probe as shown in a structural formula I in the specification. The fluorescence signal intensity of the probe on Cys is 80 times that on Hcy, 30times that on GSH and 260 times that on SO2. Cys can be highly selectively detected, and the interference of Hcy, GSH and SO2 can be distinguished at the same time. The detection limits on Hcy, GSH and SO2 are respectively 0.21 [mu]M, 0.35 [mu]M and 0.15 [mu]M, and the detection limit on Cys is as low as 13 nM. The probe is low in cytotoxicity, the fluorescence response change of the probe to Cys, Hcy, GSH and SO2 can be obviously observed through a cell imaging test, and the probe can be used for detecting in-vitro and in-vivo biological mercaptan and SO2.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a fluorescent probe with multiple active sites and a synthesis method thereof, and the probe can simultaneously differentiate and detect cysteine ​​(Cys) and homocysteine ​​(Hcy) in cells , glutathione (GSH) and sulfur dioxide (SO 2 )Applications. Background technique [0002] Small molecule thiols play very important roles in many physiological processes and are closely related to many diseases. Among them, Cys, Hcy and GSH are the most common biothiols in organisms. Cys, Hcy and GSH have similar structural features and reactivity, but they have completely different physiological functions, and many diseases are closely related to their concentration changes. On the other hand, long-term human exposure to sulfite may lead to cardiovascular-related diseases, such as ischemic heart disease, myocardial ischemia, spontaneous hypertension and hypoxic pulmo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D405/06G01N21/64
CPCC07D405/06C09K11/06C09K2211/1029C09K2211/1088G01N21/6428G01N2021/6432
Inventor 查晓明任岩申毕雪源李春正
Owner CHINA PHARM UNIV
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